Lipoic acid has been promoted as an antioxidant effective in a variety of conditions in which there is oxidant damage to the vascular bed, including diabetes mellitus and atherosclerosis. An early manifestation of such damage is dysfunction of the vascular endothelium due to deficient generation or excessive destruction of nitric oxide. Whether lipoic acid can spare nitric oxide and thus lessen endothelial dysfunction is an unanswered question with important clinical implications. We have shown that cultured human endothelial cells take up lipoic acid and reduce it to dihydrolipoic acid. The latter protects the cells in a concentration dependent manner against both internal and external oxidant stresses, and this is associated with an increase in nitric oxide generation by the cells. In this proposal we seek to confirm these novel results, to clarify the mechanism by which they occur, and to determine whether they might be relevant to clinical atherosclerosis. In the first aim we will assess the antioxidant efficacy of lipoic acid in endothelial cells in culture. Oxidant stress will be initiated by agents with biological relevance for the cells, including H202 and minimally oxidized human low density lipoprotein (LDL). Cell protection by lipoic acid will be assessed using a fluorescence-based assay of intracellular oxidant stress, by measuring lipid peroxidation as F2-isoprostanes, and by assay of cellular antioxidants. In the second aim, we will determine whether and how these antioxidant effects of lipoic acid can explain its ability to enhance endothelial nitric oxide generation. We will focus on whether intracellular dihydrolipoic acid acts directly or through glutathione, whether it is synergistic with ascorbic acid, and whether it prevents or reverses impaired nitric oxide generation due to oxidized LDL. Showing that lipoic acid enhances endothelial nitric oxide generation and action will form the basis for a small double-blind, placebo-controlled clinical trial of its ability to reverse nitric oxide-dependent endothelial dysfunction (aim 3). Subjects with increased oxidant stress due to hypercholesterolemia will be studied before and after taking lipoic acid or placebo for 8 weeks with regard to changes in plasma and urinary F2-isoprostanes and flow-mediated brachial artery dilation. Despite the widespread use of lipoic acid, these studies are necessary to establish the biological basis and rationale for the clinical use of the agent.